PROJECT SUMMARY/ABSTRACT Burkholderia mallei, the etiologic agent of glanders, causes severe disease in humans and animals and is a potential agent of biological warfare and terrorism. Studies indicate that this select agent expresses several important virulence determinants. Many of these factors, however, remain poorly defined at the molecular level. Because of this, one of the long term objectives of our research is to better understand the molecular mechanisms used by this organism to cause disease and to develop strategies to treat these diseases. Type VI secretion (T6S) is a newly characterized mechanism for protein transport that is widespread amongst Gramnegative bacteria and represents a promising target for the development of novel therapeutics. Several studies indicate that T6S systems (T6SSs) are key virulence determinants expressed by a number of important bacterial pathogens. At present, the specific function(s) of many of these systems remains unclear. It is believed, however, that T6SSs are involved in the translocation of bacterial effector proteins into eukaryotic cells. T6SSs appear to be highly regulated at the genetic level and typically involve transcriptional activator proteins to ensure expression at appropriate times. Relatively little is known about the structure and assembly of the T6S apparatus. Based on sequence homologies, however, a set of conserved T6SS core proteins have been identified. Included amongst these are the valine-glycine repeat (VgrG) proteins which have been shown to behave both as structural components of the T6SS machinery and as secreted effectors. Recently, studies have demonstrated that the cluster 1 T6SS (T6SS-1) expressed by B. mallei is required for the survival of this organism in a hamster model of glanders. Additionally, studies in our lab have shown that T6SS-1 mutants exhibit growth, actin-based motility and multinucleated giant cell formation defects in murine macrophages suggesting a critical role for T6SS-1 in the intracellular lifestyle of B. mallei. These observations provide the basis for the proposed research. We hypothesize that T6SS-1 associated effector(s) influence the intracellular behavior of B. mallei in phagocytic cells. The goal of the proposed research is to use a combination of genetic, immunological, biochemical and cellular approaches to characterize the regulation and function of T6SS-1 at the molecular level. To test our hypothesis, we have formulated the following specific aims: Specific Aim 1. Examine the intracellular behavior of B. mallei T6SS-1 mutants in murine, human and equine phagocytic cell lines. Specific Aim 2. Identify and characterize putative effector-domains associated with B. mallei VgrG1. Specific Aim 3. Identify and characterize regulatory elements required for B. mallei T6SS- 1 gene expression. Collectively, these studies will significantly increase our understanding of the contribution of T6SS-1 to the pathogenesis of disease caused by this organism as well as yield important clues for the future design of therapeutics and vaccine candidates to combat glanders.